Creatine kinase (CK) occurs in animal body fluids and tissue in the form of three known isoenzymes, designated CK-BB, CK-MM and CK-MB. Each of these three isoenzymes, namely CK-BB, CK-MM, and CK-MB, differs one from the other by virtue of containing a different combination of subunits designated M or B. CK-BB has two B subunits, CK-MM has 2 M subunits, and CK-MB has one M and one B subunit. Determining the mere presence of creatine kinase MB isoenzyme (CK-MB) in biological fluids, especially in a patient's serum, has become very useful in the diagnosis of myocardial infarction. (Galen, RS., Human Path 6: No. 2, 145-147, Apr. 1975).
A difficulty encountered in immunological methods for determining CK-MB in biological fluids is interference from CK-MM and CK-BB. Antibodies raised against CK-MM, CK-BB, or CK-MB by virtue of an identical subunit will react immunologically with CK-MB and CK-MM, with CK-MB and CK-BB, or with all three isoenzymes, respectively. Most biological fluids suspected of containing CK-MB will often contain CK-MM in an appreciable amount and CK-BB and CK-MB in a much lesser amount. As a result a problem encountered in the prior art in measuring CK-MB has been interferences from CK-BB and CK-MM.
Current methods including immunological ones for determining the presence of CK-MB and the disadvantages of such methods have recently been reported and reviewed (Current Problems in Cardiology, Vol. III, No. 12, March, 1979 p. 7-28). These methods have not been very satisfactory in resolving the difficulty of interference of CK-MM and CK-BB in the determination of CK-MB. Among these reported methods are immunological ones such as the enzyme immunoassay procedure of U.S. Pat. No. 4,067,775 wherein antibodies are used to inhibit the enzymatic activity of interfering substances and enzyme immunoprecipitin methods such as the ones described in U.S. Pat. Nos. 4,012,285 and 3,932,221 wherein CK isoenzymes are precipitated by antibodies.
The methods described in U.S. Pat. Nos. 4,012,285 and 3,932,221 employ single antibody complexes to precipitate differentially the various isoenzymes in an attempt to determine CK-MB indirectly. These methods require a determination of total CK activity before precipitation and the determination of residual CK activity after precipitation, with the difference in activities being a measure of CK-MB. The activities are determined by enzymatic assays. These methods are laborious and highly subjected to error by virtue of having to measure a difference between two quantities.